The field of this invention involves a hybridization assay system for detecting target polynucleotide sequences in deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) samples. More specifically, the invention is concerned with a method for increasing the sensitivity and where necessary the selectivity of the hybridization assay using labelled oligonucleotide probes to detect specific target sequences.
Hybridization assays may be employed for the detection and identification of DNA or RNA sequences. Published methods as used particularly in recombinant DNA research are described in Methods in Enzymology, Vol. 68, pp. 379-469 (1979); and Vol. 65, Part 1, pp. 468-478 (1968). One such method involving a preliminary separation of the nucleic acid fragments by electrophoresis is known as the "Southern Blot Filter Hybridization Method." See Southern, J. Mol. Biol. (1975) 98: 503. Hybridization probe methods for detecting pathogens are described in U.S. Pat. No. 4,358,539.
There has been a recognized need for improving hybridization assays. Instead of radiolabelled probes, which can require several days for development of the X-ray film, detection by means of luminescent-labelled probes are claimed to have advantages. (See published European patent application No. 0 070 687.) A procedure for obtaining increased sensitivity from luminescent-labelled probes is disclosed in published European patent application No. 0 070 685. Two probes are employed, having respectively a chemiluminescent catalyst and an absorber/emitter (fluorophore) moiety. Upon hybridization to the appropriate polynucleotide target sequence, the light signal from the probes is shifted to a higher wavelength. This wavelength shifting leads to an improved and more sensitive assay system.
As an alternative to employing long double-stranded probes resulting from recombinant DNA cloning procedures, short synthetically produced single-stranded probes have been described. (See, for example, PCT Publication No. WO 84/03285). Such short probes, which normally contain from 18 to 40 nucleotides, are available from Molecular Biosystems, Inc., San Diego, Calif. Typically, such short probes can be labelled with biotin, enzymes, and fluorescent reporter groups. These probes can be constructed complementary to a unique sequence of the target DNA or RNA fragments. Furthermore, they are in stable single-stranded form and renaturation cannot interfere with the assay. Short labelled DNA probes as described above, in principle, should be useful in developing rapid and convenient non-radioisotope DNA hybridization assays. With existing procedures, concentrations of the hybridized labelled probe can be detected down to about 10.sup.-16 to 10.sup.-17 moles under ideal conditions. For most of the potentially important clinical applications of DNA probes (infectious diseases, latent virus, genetic disorders, etc.), it will be necessary to have sensitivity levels of at least 10.sup.-18 moles and lower. With present techniques the inherent sensitivities possible with non-radioisotope labels such as fluorophores and enzymes are still limited by a wide variety of problems. In the case of fluorescent labels, background fluorescence and light scattering from the hybridization support materials (nitrocellulose and nylon filters, polystyrene beads, etc.) lead to significant loss of inherent sensitivity. In the case of enzyme labels, non-specific binding of the labeled probe and interfering stains and colors from sample material can limit ultimate sensitivity of this label. Other problems superimposed upon those metioned above lead to further losses of sensitivity and limit the practicality of these systems for clinical diagnostic purposes.